Find all perfect matches for short sequences to human reference genome

Question

[Mick]

Hi guys,

I'm trying to get something done that sound super simple but I'm stuck and I don't know how to go about it.

I have a large excel file with Primer sequences that someone designed for the human genome and I need to find out which part of the genome they cover. All i have is the sequence for the forward and reverse primer. So ideally I'd like to align the forward and reverse primer to the genome, I only want perfect matches to the genome because the primers have been designed in such a way.

There should be a linux tool to do this right? Maybe Blast, or bowtie? If anyone could guide me in the right direction I would much appreciate that.

Thanks :)

[jean.elbers]

bbmap has the following modes that might be of use...

perfectmode=f           Allow only perfect mappings when set to true (very fast).
semiperfectmode=f       Allow only perfect and semiperfect (perfect except for N's in the reference) mappings.

[ATpoint]

bowtie is perfectly fine. I would set all mismatch and penalty parameters to 1000, set seed mismatches to zero etc. to ensure only perfect matches are returned.

[Mick]

Thank you for the quick reply, I'll let you know how it works :)

[ATpoint]

For bowtie I think one could use -n 0 -k 1 -m 1 --best --strata -v0. For bowtie2 I used in the past (but this was for NGS perfect matches not short primers) --end-to-end -N 0 --mp 10000 --np 10000 --rdg 10000 --rfg 10000. Try things out a bit :)

[GenoMax]

Depending on how many sequences there are just use in silico PCR tool: http://genome.ucsc.edu/cgi-bin/hgPcr?db=hg38.
You can also use the web interface for blat for a more flexible search. http://genome.ucsc.edu/cgi-bin/hgBlat?command=start

Answer 1

[Mick]

Thank you guys so much for all the replys. I tried bowtie and used these parameters:

bowtie hg19 -v 0 -a -X 800 -r1 "sequences1.txt" -r2 "sequences2.txt"

It worked really well for the most part. It only missed a couple of primers, where it didn't find any alignment. I manually searched for the location of these primers with blast. They were perfect matches to the genome, I don't really know why bowtie may have missed those. But anyhow, thank you guys so much for the help. :)